Interpretive Summary: The soybean aphid is a significant soybean pest, yet gene expression and functional genomics studies of the aphid are hindered by a lack of stable reference genes. Quantitative real-time PCR (qRT-PCR) is a common tool for quantifying messenger RNA transcripts. To normalize results, a reference gene is mandatory. We evaluated 7 potential reference genes (SDFA, succinate dehydrogenase flavoprotein subunit a, EF1a, elongation factor-1 alpha, HEL, Helicase, GAPDH, glyceraldehyde-3 phosphate dehydrogenase, RPS9, ribosomal protein S9, TBP, TATA-box binding protein, and UBQ, ubiquitin conjugating protein) to determine the most efficient reference genes that have stable expression among tissues, developmental stages and aphids fed on susceptible and host-plant resistant plants. Stability was determined and the results revealed high stability of TBP compared to the other reference genes profiled across all samples. Stable expression was shown with RPS9 among aphids on susceptible and resistant plants and tissues and aphid developmental stages. Therefore, we recommend the gene TBP as a suitable reference gene for normalization of soybean aphid gene expression studies. Additionally, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate normalization of quantitative real-time PCR data in the soybean aphid.

Technical Abstract:
Quantitative real-time PCR (qRT-PCR) is a common tool for quantifying mRNA transcripts. To normalize results, a reference gene is mandatory. Aphis glycines is a significant soybean pest, yet gene expression and functional genomics studies are hindered by a lack of stable reference genes. We evaluated 7 potential reference genes (SDFA, succinate dehydrogenase flavoprotein subunit a, EF1a, elongation factor-1 alpha, HEL, Helicase, GAPDH, glyceraldehyde-3 phosphate dehydrogenase, RPS9, ribosomal protein S9, TBP, TATA-box binding protein, and UBQ, ubiquitin conjugating protein) to determine the most efficient reference genes that have stable expression among tissues, developmental stages and aphids fed on susceptible and host-plant resistant plants. Stability was determined using GeNorm and NormFinder. Results revealed high stability of TBP compared to the other reference genes profiled across all samples. Stable expression was shown with RPS9 among aphids on susceptible and resistant plants and EF1a tissues and developmental stages. Therefore, we recommend the gene TBP as a suitable reference gene for normalization of A. glycines gene expression studies. Additionally, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate normalization of qRT-PCR data in A. glycines.